The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a central role in many cellular responses to Ca2+-mobilization and is implicated in many aspects of health and disease. It is most abundant in brain where it regulates synaptic transmission in both the pre- and post-synaptic cells, and is implicated in learning and memory. The long-term aim is to understand mechanisms regulating phosphorylation of targeted substrates by CaMKII. Three related aspects of this aim are addressed: The mechanism by which Ca2+-independent autophosphorylation of CaMKII blocks interaction with calmodulin (CaM) and desensitizes the kinase to Ca2+/CaM will be investigated. A cDNA encoding the alpha subunit of mouse brain CaMKII will be mutated at positions corresponding to the known Ca2+-independent autophosphorylation sites (Thr3O5, Thr3O6 and Ser3l4) and expressed in insect cells using baculovirus. CaM-binding and autophosphorylation properties of mutant kinases will be characterized. These studies will be important in understanding the biochemical mechanisms involved in Ca2+-independent autophosphorylation as well as its physiological role. The phosphorylation state of CaMKII autophosphorylation sites depends on the rates of both phosphorylation and dephosphorylation. Therefore, protein phosphatases involved in dephosphorylating CAMKII will be identified. CaMKII will be [32P]-labeled at either Thr286, the Ca2+/CaM-dependent site responsible for the partially Ca2+-independent form of CaMKII, or at specific Ca2+-independent sites (Thr305/Thr306 or Ser3l4) and used as a substrate for neuronal protein phosphatases. Activities will be attributed to either known or novel enzymes by using a series of specific activators and inhibitors, and also by their chromatographic behavior. Characterization of the site specificity, identity and relative abundance of protein phosphatases acting on CaMKII is essential for a complete understanding of CaMKII regulation in vivo. Substrates phosphorylated in response to elevated Ca2+ in vivo may be targeted in part by co-localization of CaMKII with its substrates, in part on to regulation of kinase activity. The location of CaMKII may also affect its regulation by Ca2+ and phosphatases. The role of specific binding proteins in determining the subcellular localization of CaMKII will be investigated. CaMKII-binding proteins will be identified in rat tissue extracts and the possible regulation of binding by phosphorylation and other factors will be investigated.